1. Field of the Invention (Technical Field)
The present invention relates to coatings and methods of use of non-thrombogenic compositions for selectively inhibiting and promoting cellular attachment, including a silyl-heparin-fibronection composition for promoting cellular attachment.
2. Background Art
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-a-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Heparin is naturally present in various tissues, including liver and lung, as well as the luminal surface of endothelial cells. It is composed of repeating units of D-glucuronic acid and D-glucosamine, both sulfated, in a 1,4-xcex1 linkage. Heparin is an anticoagulant, and it has been reported that on the surface of endothelial cells heparin minimizes fibrin accumulation. When administered as a parenteral drug, heparin activates anti-thrombin III, which leads to inactivation of thrombin and ultimately systemic inhibition of fibrin formation.
A number of medical devices that come in contact with blood have been coated with heparin with the goal of taking advantage of its thrombo-resistant nature. Stents, catheters, oxygenator fibers, and cardiac bypass circuits are examples of medical devices that have been coated with heparin (Niimi et al., Anesth Analg 89:573-9, 1999; lnui et al., Artif Organs, 23:1107-12, 1999). Various strategies have been developed to attach heparin to medical polymer surfaces including chemical conjugation (Siefert et al., J Biomater Sci Polym Ed, 7:277-87, 1995), plasma glow discharge methods (Kim et al., Biomaterials, 21:121-30, 2000), the combination of both, and hydrophobic interaction as described herein (U.S. Pat. No. 5,955,588).
Heparin has a number of other biological actions related to its presence in the extracellular matrix. In the extracellular matrix, heparin and its chemical relative heparan sulfate is complexed into a scaffolding onto which cells attach. In this scaffolding, heparin is bound by fibronectin and other adhesive molecules, which in turn bind to cells. Extracellular matrix heparin and heparin sulfate also act as reservoirs for growth factors, not only binding growth factors but also protecting them from protease degradation. Fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and bone morphogenic protein (BMP) are examples of growth factors that complex to heparin.
The ability of heparin to bind adhesive molecules and growth factors has lead to a number of efforts to use heparin complexes to improve implantable medical device surfaces by providing surfaces to which cells can attach and migrate. Other researchers have explored direct coatings of fibronectin, and peptides and peptide mimetics derived from fibronectin, with the goal of increasing cell attachment (Walluscheck et al., Eur J Vasc Endovasc Surg, 12:321-30, 1996; Boxus et al., J Bioorg Med Chem, 6:1577-95,1998; Tweden et al., J. Heart Valve Dis, 4 Suppl 1:S90-7, 1995). Vascular grafts, for example, would be improved by a surface that supports the growth of endothelial cells. Current vascular grafts of polytetrafluoroethylene and polyethylene terephthalate do not support endothelization, and consequently patients must be maintained on long-term anti-platelet therapy.
Fibronectins function as adhesive, ligand molecules interacting with specific receptors on the cell surface. Cells types that attach to fibronectin include fibroblasts, endothelial cells, smooth muscle cells, osteoblasts, and chondrocytes.
Other investigators have used heparin/fibronectin complexes to provide cell adhesion to polymeric surfaces. For example, heparin-albumin conjugates have been immobilized on carbon dioxide gas plasma-treated polystyrene (Bos et al., J. Biomed Mater Res, 47:279-91, 1999) and complexed to fibronectin. The fibronectin on these surfaces increased the attachment of endothelial cells. Bos et al. (Tissue Eng 4:267-79, 1998; J Biomed Mater Res,47:279-91, 1999) reported that endothelial cells grew to confluency on CO2 gas plasma-treated polystyrene coated with an albumin-heparin conjugate. Ishihara et al. (J Biomed Mater Res, 50: 144-152, 2000) reported that a heparin-conjugated polystyrene promoted cell attachment of fibroblasts, smooth muscle cell and endothelial cells. The fibroblasts grown on heparin-conjugated polystyrene had growth rates at least comparable to fibronectin-coated, gelatin-coated, or tissue culture-treated media.
A simple method of efficiently complexing fibronectin or other adhesive molecules, including derivatives or mimics of the foregoing, to a heparin complex would have wide applicability for attaching cells to prostheses, including vascular grafts, bone and cartilage implants, nerve guides and the like. Particularly needed is a method and composition permitting use of a wide variety of adhesive molecules, including fibronectin, laminin and the like, as part of a coating for implantable medical devices. There remains a need in the art for coating compositions for implantable medical devices that promote cellular attachment, and further wherein cellular attachment can be modulated by the quantity of adhesive molecule, and which can be applied simply and easily with no specialized equipment or techniques.
The present invention provides an amphiphatic cell-attachment coating composition for medical device surfaces, which composition includes a covalent complex of from 1 to 30 hydrophobic silyl moieties of Formula I: 
wherein
R1 is an C1-18 alkyl or C6-32 aryl group,
each R2 is independently selected from the group consisting of C1-18 alkyl and C6-32 aryl
R3 is N or O, and
n is a number from 1 to 10
directly bound to sodium heparin via covalent bonding, with an adhesive molecule directly bound to the sodium heparin. The hydrophobic silyl moieties may be bound to the surfaces via hydrophobic bonding interactions. Further, the complex can include from 2 to 25 hydrophobic silyl moieties covalently bound to one heparin molecule. In Formula I, R1 can be benzyl and R2 can be an alkyl. In a preferred embodiment, the complex is [benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or [benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. In a preferred embodiment, the adhesive molecule is fibronectin; in alternative embodiments, the adhesive molecule may be laminin, vitronectin, thrombospondin, gelatin, polylysine, polyornithine, peptide polymers containing adhesive sequences and heparin binding sequences, sulfated complex carbohydrates, dextran sulfate, growth hormones, cytokines, lectins, or peptidic polymers thereof.
The invention further provides a non-thrombogenic medical device for cellular attachment, including surfaces for contacting blood, which surfaces have coated thereon an non-thrombogenic coating composition comprising a covalent complex of from 1 to 30 hydrophobic silyl moieties of Formula I: 
wherein
R1 is an C1-18 alkyl or C6-32 aryl group,
each R2 is independently selected from the group consisting of C1-18 alkyl and C6-32 aryl,
R3 is N or O, and
n is a number from 1 to 10
directly bound to heparin via covalent bonding, with an adhesive molecule directly bound to the heparin. The hydrophobic silyl moieties may be bound to the coated surfaces via hydrophobic bonding interactions. Further, the complex can include from 2 to 25 hydrophobic silyl moieties covalently bound to one heparin molecule. In Formula I, R1 can be benzyl and R2 can be an alkyl. In a preferred embodiment, the complex is [benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or [benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. In a preferred embodiment, the adhesive molecule is fibronectin; in alternative embodiments, the adhesive molecule may be laminin, vitronectin, thrombospondin, gelatin, polylysine, polyornithine, peptide polymers containing adhesive sequences and heparin binding sequences, sulfated complex carbohydrates, dextran sulfate, growth hormones, cytokines, lectins, or peptidic polymers thereof. The devices of this invention include blood gas exchange devices, blood filters, artificial blood vessels, artificial valves, prosthetics, blood shunts, catheters, bone replacements, cartilage replacements and nerve growth guides.
In yet another embodiment, the invention provides a method for rendering a tissue- or blood-contacting surfaces of a medical device resistant to fibrin accumulation while promoting cellular adhesion, which method includes coating the surfaces with an non-thrombogenic coating composition comprising a covalent complex of from 1 to 30 hydrophobic silyl moieties of Formula I: 
wherein
R1 is an C1-18 alkyl or C6-32 aryl group,
each R2 is independently selected from the group consisting of C1-18 alkyl and C6-32aryl,
R3 is N or O, and
n is a number from 1 to 10
directly bound to heparin via covalent bonding, and attaching to the heparin an adhesive molecule. In this method, the hydrophobic silyl moieties can be bound to the surfaces via hydrophobic bonding interactions. From 2 to 25 hydrophobic silyl moieties can be covalently bound to one heparin molecule. In a preferred embodiment, R1 is benzyl and R2 is an alkyl. In preferred embodiments, the complex is [benzyl-bis(dimethylsilylmethyl)]-(N-heparinyl)-carbamate or [benzyl-tris(dimethylsilylmethyl)]-(N-heparinyl)-carbamate. The method can further include the step of solubilizing the complex in a solvent prior to the step of coating the surface. In one embodiment, the step of coating the surfaces includes dipping the surface into the coating composition including the complex. In another embodiment, the step of coating the surface includes pumping the coating composition including the complex onto the surface. In a preferred embodiment of this method, the adhesive molecule is fibronectin; in alternative embodiments, the adhesive molecule may be laminin, vitronectin, thrombospondin, gelatin, polylysine, polyornithine, peptide polymers containing adhesive sequences and heparin binding sequences, sulfated complex carbohydrates, dextran sulfate, growth hormones, cytokines, lectins, or peptidic polymers thereof. The method can also include the step of solubilizing the adhesive molecule in a solvent prior to the step of attaching the adhesive molecule. The step of attaching the adhesive molecule includes dipping the surface coated with the complex into a solubilized adhesive molecule composition. Alternatively, the step of attaching the adhesive molecule includes pumping the solubilized adhesive molecule composition onto a surface coated with the complex.
A primary object of the present invention is to provide an amphipathic heparin-fibronectin coating composition for implantable medical devices, which promotes cellular attachment.
A further objective of the invention is to provide a coating the composition of which can be varied, such that absent an adhesion molecule the coating inhibits fibrin deposition, but when the coating includes an adhesion molecule, the coating promotes cellular attachment and cell growth.
A further object of the invention is to provide coating compositions utilizing fibronectin, derivations of fibronectin, peptide mimics of fibronectin, laminin, vitronectin, thrombospondin, gelatin, collagen and subtypes thereof, gelatin, polylysine, polyornithine, and other adhesive molecules or derivatives or mimics of other adhesive molecules.
A further object of the present invention is to provide a cost effective and commercially feasible method for coating polymeric medical devices, including biodegradable medical devices, with a non-thrombogenic coating that inhibits cell attachment.
A further object of the present invention is to provide a cost effective and commercially feasible method for coating polymeric medical devices, including biodegradable medical devices, with a coating that resists fibrin accumulation and promotes cell attachment and growth utilizing a complex with an adhesive molecule, including fibronectin, peptide mimics of fibronectin, laminin, vitronectin, thrombospondin, gelatin, collagen and subtypes thereof, gelatin, polylysine, polyornithine, and other adhesive molecules or derivatives or mimics of other adhesive molecules.
A primary advantage of the present invention is that it provides for coating medical devices of complex geometries and surfaces with a durable and low-cost coating that promotes uniform cell growth and attachment.
Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.